EGVI endoglucanase and nucleic acids encoding the same

ABSTRACT

The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

GOVERNMENT SUPPORT

Portions of this work were funded by Subcontract No. ZCO-30017-01 withthe National Renewable Energy Laboratory under Prime Contract No.DE-AC36-99GO10337 with the U.S. Department of Energy. Accordingly, theUnited States Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to isolated egl6 nucleic acid sequenceswhich encode polypeptides having endoglucanase activity. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the nucleic acid sequences as well as methods for producingrecombinant EGVI polypeptides.

REFERENCES

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BACKGROUND OF THE INVENTION

Cellulose and hemicellulose are the most abundant plant materialsproduced by photosynthesis. They can be degraded and used as an energysource by numerous microorganisms, including bacteria, yeast and fungi,that produce extracellular enzymes capable of hydrolysis of thepolymeric substrates to monomeric sugars (Aro et al., 2001). As thelimits of non-renewable resources approach, the potential of celluloseto become a major renewable energy resource is enormous (Krishna et al.,2001). The effective utilization of cellulose through biologicalprocesses is one approach to overcoming the shortage of foods, feeds,and fuels (Ohmiya et al., 1997).

Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or betaD-glucosidic linkages) resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like. Cellulases have beentraditionally divided into three major classes: endoglucanases (EC3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91)(“CBH”) and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC3.2.1.21) (“BG”). (Knowles et al., 1987; Shulein, 1988). Endoglucanasesact mainly on the amorphous parts of the cellulose fibre, whereascellobiohydrolases are also able to degrade crystalline cellulose(Nevalainen and Penttila, 1995). Thus, the presence of acellobiohydrolase in a cellulase system is required for efficientsolubilization of crystalline cellulose (Suumakki, et al. 2000).Beta-glucosidase acts to liberate D-glucose units from cellobiose,cello-oligosaccharides, and other glucosides (Freer, 1993).

Cellulases are known to be produced by a large number of bacteria, yeastand fungi. Certain fungi produce a complete cellulase system capable ofdegrading crystalline forms of cellulose, such that the cellulases arereadily produced in large quantities via fermentation. Filamentous fungiplay a special role since many yeast, such as Saccharomyces cerevisiae,lack the ability to hydrolyze cellulose. See, e.g., Aro et al., 2001;Aubert et al., 1988; Wood et al., 1988, and Coughlan, et al.

The fungal cellulase classifications of CBH, EG and BG can be furtherexpanded to include multiple components within each classification. Forexample, multiple CBHs, EGs and BGs have been isolated from a variety offungal sources including Trichoderma reesei which contains known genesfor 2 CBHs, i.e., CBH I and CBH II, at least 5 EGs, i.e., EG I, EG II,EG III, EGIV and EGV, and at least 2 BGs, i.e., BG1 and BG2.

In order to efficiently convert crystalline cellulose to glucose thecomplete cellulase system comprising components from each of the CBH, EGand BG classifications is required, with isolated components lesseffective in hydrolyzing crystalline cellulose (Filho et al, 1996). Asynergistic relationship has been observed between cellulase componentsfrom different classifications. In particular, the EG-type cellulasesand CBH-type cellulases synergistically interact to more efficientlydegrade cellulose. See, e.g., Wood, 1985.

Cellulases are known in the art to be useful in the treatment oftextiles for the purposes of enhancing the cleaning ability of detergentcompositions, for use as a softening agent, for improving the feel andappearance of cotton fabrics, and the like (Kumar et al., 1997).

Cellulase-containing detergent compositions with improved cleaningperformance (U.S. Pat. No. 4,435,307; GB App. Nos. 2,095,275 and2,094,826) and for use in the treatment of fabric to improve the feeland appearance of the textile (U.S. Pat. Nos. 5,648,263, 5,691,178, and5,776,757; GB App. No. 1,358,599; The Shizuoka Prefectural HamamatsuTextile Industrial Research Institute Report, Vol. 24, pp. 54-61, 1986),have been described.

Hence, cellulases produced in fungi and bacteria have receivedsignificant attention. In particular, fermentation of Trichoderma spp.(e.g., Trichoderma longibrachiatum or Trichoderma reesei) has been shownto produce a complete cellulase system capable of degrading crystallineforms of cellulose. U.S. Pat. No. 5,475,101 discloses the purificationand molecular cloning of one particularly useful enzyme designated EGIIIwhich is derived from Trichoderma longibrachiatum.

Although cellulase compositions have been previously described, thereremains a need for new and improved cellulase compositions for use inhousehold detergents, stonewashing compositions or laundry detergents,etc. Cellulases that exhibit resistance to surfactants (e.g., linearalkyl sulfonates, LAS), improved performance under conditions of thermalstress, increased or decreased cellulolytic capacity, and/or high levelexpression in vitro, are of particular interest.

SUMMARY OF THE INVENTION

The invention provides an isolated cellulase protein, identified hereinas EGVI, and nucleic acids which encode EGVI.

In one aspect, EGVI polypeptides or proteins comprise a sequence havingat least 80%, 85%, 90%, 95%, 98% or more sequence identity to thesequence presented as SEQ ID NO:2.

In a related aspect, the invention includes (i) fragments of EGVI,preferably at least about 20-100 amino acids in length, more preferablyabout 100-200 amino acids in length, and (ii) a pharmaceuticalcomposition comprising EGVI. In various embodiments, the fragmentcorresponds to the N-terminal domain of EGVI or the C-terminal domain ofEGVI.

In another aspect the invention includes an isolated polynucleotidehaving a sequence which encodes EGVI, a sequence complementary to theegl6 coding sequence, and a composition comprising the polynucleotide.The polynucleotide may be mRNA, DNA, cDNA, genomic DNA, or an antisenseanalog thereof.

An egl6 polynucleotide may comprise an isolated nucleic acid moleculewhich hybridizes to the complement of the nucleic acid presented as SEQID NO: 1 under moderate to high stringency conditions, where the nucleicacid molecule encodes an EGVI polypeptide that exhibits endoglucanaseactivity.

The polynucleotide may encode an EGVI protein having at least 80%, 85%,90%, 95%, 98% or more sequence identity to the sequence presented as SEQID NO:1. In a specific embodiment, the polynucleotide comprises asequence substantially identical to SEQ ID NO:1. The invention alsocontemplates fragments of the polynucleotide, preferably at least about15-30 nucleotides in length.

The invention further provides recombinant expression vectors containinga nucleic acid sequence encoding EGVI or a fragment or splice variantthereof, operably linked to regulatory elements effective for expressionof the protein in a selected host. In a related aspect, the inventionincludes a host cell containing the vector.

The invention further includes a method for producing EGVI byrecombinant techniques, by culturing recombinant prokaryotic oreukaryotic host cells comprising nucleic acid sequence encoding EGVIunder conditions effective to promote expression of the protein, andsubsequent recovery of the protein from the host cell or the cellculture medium.

In yet another aspect, the invention includes an antibody specificallyimmunoreactive with EGVI.

Analytical methods for detecting egl6 nucleic acids and EGVI proteinsalso form part of the invention.

In another aspect the invention provides for an enzymatic compositionuseful in the conversion of cellulose to sugars and/or ethanol. In apreferred embodiment the enzymatic composition comprises EGVI. Thecomposition may further comprise additional cellulase enzymes such asother endoglucanases, beta-glucosidases and/or cellbiohydrolases. Thecomposition may be enriched in EGVI.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a single stranded depiction of the nucleic acid sequence (SEQID NO:1), of the T. reesei egl6 cDNA, wherein the non-coding sequence isindicated as bolded.

FIG. 2 shows the predicted amino acid sequence (SEQ ID NO:2) and signalsequence (SEQ ID NO:3) based on the nucleotide sequence provided in FIG.1, wherein the signal sequence is indicated as bolded.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions.

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al., 1989, and Ausubel F M et al., 1993, for definitions andterms of the art. It is to be understood that this invention is notlimited to the particular methodology, protocols, and reagentsdescribed, as these may vary.

All publications cited herein are expressly incorporated herein byreference for the purpose of describing and disclosing compositions andmethodologies which might be used in connection with the invention.

The term “polypeptide” as used herein refers to a compound made up of asingle chain of amino acid residues linked by peptide bonds. The term“protein” as used herein may be synonymous with the term “polypeptide”or may refer, in addition, to a complex of two or more polypeptides.

The term “nucleic acid molecule” includes RNA, DNA and cDNA molecules.It will be understood that, as a result of the degeneracy of the geneticcode, a multitude of nucleotide sequences encoding a given protein suchas EGVI may be produced. The present invention contemplates everypossible variant nucleotide sequence, encoding EGVI, all of which arepossible given the degeneracy of the genetic code.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence which is not native to the cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,transformation, microinjection, electroporation, or the like. A“heterologous” nucleic acid construct may contain a control sequence/DNAcoding sequence combination that is the same as, or different from acontrol sequence/DNA coding sequence combination found in the nativecell.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

Accordingly, an “expression cassette” or “expression vector” is anucleic acid construct generated recombinantly or synthetically, with aseries of specified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid sequence to betranscribed and a promoter.

As used herein, the term “plasmid” refers to a circular double-stranded(ds) DNA construct used as a cloning vector, and which forms anextrachromosomal self-replicating genetic element in many bacteria andsome eukaryotes.

As used herein, the term “selectable marker-encoding nucleotidesequence” refers to a nucleotide sequence which is capable of expressionin cells and where expression of the selectable marker confers to cellscontaining the expressed gene the ability to grow in the presence of acorresponding selective agent, or under corresponding selective growthconditions.

As used herein, the term “promoter” refers to a nucleic acid sequencethat functions to direct transcription of a downstream gene. Thepromoter will generally be appropriate to the host cell in which thetarget gene is being expressed. The promoter together with othertranscriptional and translational regulatory nucleic acid sequences(also termed “control sequences”) are necessary to express a given gene.In general, the transcriptional and translational regulatory sequencesinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences.

“Chimeric gene” or “heterologous nucleic acid construct”, as definedherein refers to a non-native gene (i.e., one that has been introducedinto a host) that may be composed of parts of different genes, includingregulatory elements. A chimeric gene construct for transformation of ahost cell is typically composed of a transcriptional regulatory region(promoter) operably linked to a heterologous protein coding sequence,or, in a selectable marker chimeric gene, to a selectable marker geneencoding a protein conferring antibiotic resistance to transformedcells. A typical chimeric gene of the present invention, fortransformation into a host cell, includes a transcriptional regulatoryregion that is constitutive or inducible, a protein coding sequence, anda terminator sequence. A chimeric gene construct may also include asecond DNA sequence encoding a signal peptide if secretion of the targetprotein is desired.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNAencoding a secretory leader is operably linked to DNA for a polypeptideif it is expressed as a preprotein that participates in the secretion ofthe polypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. Generally, “operably linked” means thatthe DNA sequences being linked are contiguous, and, in the case of asecretory leader, contiguous and in reading frame. However, enhancers donot have to be contiguous.

Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors, linkersor primers for PCR are used in accordance with conventional practice.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, that may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons).

In general, nucleic acid molecules which encode EGVI or an analog orhomologue thereof will hybridize, under moderate to high stringencyconditions to the sequence provided herein as SEQ ID NO:1. However, insome cases an EGVI-encoding nucleotide sequence is employed thatpossesses a substantially different codon usage, while the proteinencoded by the EGVI-encoding nucleotide sequence has the same orsubstantially the same amino acid sequence as the native protein. Forexample, the coding sequence may be modified to facilitate fasterexpression of EGVI in a particular prokaryotic or eukaryotic expressionsystem, in accordance with the frequency with which a particular codonis utilized by the host. Te'o, et al. (2000), for example, describes theoptimization of genes for expression in filamentous fungi.

A nucleic acid sequence is considered to be “selectively hybridizable”to a reference nucleic acid sequence if the two sequences specificallyhybridize to one another under moderate to high stringency hybridizationand wash conditions. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe. Forexample, “maximum stringency” typically occurs at about Tm-5° C. (5°below the Tm of the probe); “high stringency” at about 5-10° below theTm; “intermediate stringency” at about 10-20° below the Tm of the probe;and “low stringency” at about 20-25° below the Tm. Functionally, maximumstringency conditions may be used to identify sequences having strictidentity or near-strict identity with the hybridization probe; whilehigh stringency conditions are used to identify sequences having about80% or more sequence identity with the probe.

Moderate and high stringency hybridization conditions are well known inthe art (see, for example, Sambrook, et al., 1989, Chapters 9 and 11,and in Ausubel, F. M., et al., 1993, expressly incorporated by referenceherein). An example of high stringency conditions includes hybridizationat about 42° C. in 50% formamide, 5×SSC, 5× Denhardt's solution, 0.5%SDS and 100 μg/ml denatured carrier DNA followed by washing two times in2×SSC and 0.5% SDS at room temperature and two additional times in0.1×SSC and 0.5% SDS at 42° C.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the terms “transformed”, “stably transformed” or“transgenic” with reference to a cell means the cell has a non-native(heterologous) nucleic acid sequence integrated into its genome or as anepisomal plasmid that is maintained through multiple generations.

As used herein, the term “expression” refers to the process by which apolypeptide is produced based on the nucleic acid sequence of a gene.The process includes both transcription and translation.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

It follows that the term “EGVI expression” refers to transcription andtranslation of the egl6 gene, the products of which include precursorRNA, mRNA, polypeptide, post-translationally processed polypeptides, andderivatives thereof, including EGVI from related species such asTrichoderma longibrachiatum (reesei), Trichoderma viride, Trichodermakoningii, Hypocrea jecorina and Hypocrea schweinitzii. By way ofexample, assays for EGVI expression include Western blot for EGVIprotein, Northern blot analysis and reverse transcriptase polymerasechain reaction (RT-PCR) assays for EGVI mRNA, and endoglucanase activityassays as described in Shoemaker S. P. and Brown R. D. Jr. (Biochim.Biophys. Acta, 1978, 523:133-146) and Schulein (1988).

The term “alternative splicing” refers to the process whereby multiplepolypeptide isoforms are generated from a single gene, and involves thesplicing together of nonconsecutive exons during the processing of some,but not all, transcripts of the gene. Thus a particular exon may beconnected to any one of several alternative exons to form messengerRNAs. The alternatively-spliced mRNAs produce polypeptides (“splicevariants”) in which some parts are common while other parts aredifferent.

The term “signal sequence” refers to a sequence of amino acids at theN-terminal portion of a protein, which facilitates the secretion of themature form of the protein outside the cell. The mature form of theextracellular protein lacks the signal sequence, which is cleaved offduring the secretion process.

By the term “host cell” is meant a cell that contains a vector andsupports the replication, and/or transcription or transcription andtranslation (expression) of the expression construct. Host cells for usein the present invention can be prokaryotic cells, such as E. coli, oreukaryotic cells such as yeast, plant, insect, amphibian, or mammaliancells. In general, host cells are filamentous fungi.

The term “filamentous fungi” means any and all filamentous fungirecognized by those of skill in the art. A preferred fungus is selectedfrom the group consisting of Aspergillus, Trichoderma, Fusarium,Chrysosporium, Penicillium, Humicola, Neurospora, or alternative sexualforms thereof such as Emericella, Hypocrea.

The term “cellooligosaccharide” refers to oligosaccharide groupscontaining from 2-8 glucose units and having β-1,4 linkages, e.g.,cellobiose.

The term “cellulase” refers to a category of enzymes capable ofhydrolyzing cellulose polymers to shorter cello-oligosaccharideoligomers, cellobiose and/or glucose. Numerous examples of cellulases,such as exoglucanases, exocellobiohydrolases, endoglucanases, andglucosidases have been obtained from cellulolytic organisms,particularly including fungi, plants and bacteria.

The term “cellulose binding domain” as used herein refers to portion ofthe amino acid sequence of a cellulase or a region of the enzyme that isinvolved in the cellulose binding activity of a cellulase or derivativethereof. Cellulose binding domains generally function by non-covalentlybinding the cellulase to cellulose, a cellulose derivative or otherpolysaccharide equivalent thereof. Cellulose binding domains permit orfacilitate hydrolysis of cellulose fibers by the structurally distinctcatalytic core region, and typically function independent of thecatalytic core. Thus, a cellulose binding domain will not possess thesignificant hydrolytic activity attributable to a catalytic core. Inother words, a cellulose binding domain is a structural element of thecellulase enzyme protein tertiary structure that is distinct from thestructural element which possesses catalytic activity.

As used herein, the term “surfactant” refers to any compound generallyrecognized in the art as having surface active qualities. Thus, forexample, surfactants comprise anionic, cationic and nonionic surfactantssuch as those commonly found in detergents. Anionic surfactants includelinear or branched alkylbenzenesulfonates; alkyl or alkenyl ethersulfates having linear or branched alkyl groups or alkenyl groups; alkylor alkenyl sulfates; olefinsulfonates; and alkanesulfonates. Ampholyticsurfactants include quaternary ammonium salt sulfonates, andbetaine-type ampholytic surfactants. Such ampholytic surfactants haveboth the positive and negative charged groups in the same molecule.Nonionic surfactants may comprise polyoxyalkylene ethers, as well ashigher fatty acid alkanolamides or alkylene oxide adduct thereof, fattyacid glycerine monoesters, and the like.

As used herein, the term “cellulose containing fabric” refers to anysewn or unsewn fabrics, yarns or fibers made of cotton or non-cottoncontaining cellulose or cotton or non-cotton containing cellulose blendsincluding natural cellulosics and manmade cellulosics (such as jute,flax, ramie, rayon, and lyocell).

As used herein, the term “cotton-containing fabric” refers to sewn orunsewn fabrics, yarns or fibers made of pure cotton or cotton blendsincluding cotton woven fabrics, cotton knits, cotton denims, cottonyarns, raw cotton and the like.

As used herein, the term “stonewashing composition” refers to aformulation for use in stonewashing cellulose containing fabrics.Stonewashing compositions are used to modify cellulose containingfabrics prior to sale, i.e., during the manufacturing process. Incontrast, detergent compositions are intended for the cleaning of soiledgarments and are not used during the manufacturing process.

As used herein, the term “detergent composition” refers to a mixturewhich is intended for use in a wash medium for the laundering of soiledcellulose containing fabrics. In the context of the present invention,such compositions may include, in addition to cellulases andsurfactants, additional hydrolytic enzymes, builders, bleaching agents,bleach activators, bluing agents and fluorescent dyes, cakinginhibitors, masking agents, cellulase activators, antioxidants, andsolubilizers.

As used herein, the term “decrease or elimination in expression of theegl6 gene” means that either that the egl6 gene has been deleted fromthe genome and therefore cannot be expressed by the recombinant hostmicroorganism; or that the egl6 gene has been modified such that afunctional EGVI enzyme is not produced by the recombinant hostmicroorganism.

The term “altered egl6” or “altered egl6 gene” means that the nucleicacid sequence of the gene has been altered by removing, adding, and/ormanipulating the coding sequence or the amino acid sequence of theexpressed protein has been modified.

As used herein, the term “purifying” generally refers to subjectingtransgenic nucleic acid or protein containing cells to biochemicalpurification and/or column chromatography.

As used herein, the terms “active” and “biologically active” refer to abiological activity associated with a particular protein, such as theenzymatic activity associated with a protease. It follows that thebiological activity of a given protein refers to any biological activitytypically attributed to that protein by those of skill in the art.

As used herein, the term “enriched” means that the EGVI is found in aconcentration that is greater relative to the EGVI concentration foundin a wild-type, or naturally occurring, fungal cellulase composition.The terms enriched, elevated and enhanced may be used interchangeablyherein.

A wild type fungal cellulase composition is one produced by a naturallyoccurring fungal source and which comprises one or more BGL, CBH and EGcomponents wherein each of these components is found at the ratioproduced by the fungal source. Thus, an enriched EGVI composition wouldhave EGVI at an altered ratio wherein the ratio of EGVI to othercellulase components (i.e., CBHs, beta-glucosidases and otherendoglucanases) is elevated. This ratio may be increased by eitherincreasing EGVI or decreasing (or eliminating) at least one othercomponent by any means known in the art.

Thus, to illustrate, a naturally occurring cellulase system may bepurified into substantially pure components by recognized separationtechniques well published in the literature, including ion exchangechromatography at a suitable pH, affinity chromatography, size exclusionand the like. For example, in ion exchange chromatography (usually anionexchange chromatography), it is possible to separate the cellulasecomponents by eluting with a pH gradient, or a salt gradient, or both apH and a salt gradient. The purified EGVI may then be added to theenzymatic solution resulting in an enriched EGVI solution. It is alsopossible to elevate the amount of EGVI produced by a microbe usingmolecular genetics methods to overexpress the gene encoding EGVI,possibly in conjunction with deletion of one or more genes encodingother cellulases.

Fungal cellulases may contain more than one EG component. The differentcomponents generally have different isoelectric points which allow fortheir separation via ion exchange chromatography and the like. Either asingle EG component or a combination of EG components may be employed inan enzymatic solution.

When employed in enzymatic solutions, the EG component is generallyadded in an amount sufficient to allow the highest rate of release ofsoluble sugars from the biomass. The amount of EG component addeddepends upon the type of biomass to be saccharified, which can bereadily determined by the skilled artisan. However, when employed, theweight percent of the EGVIII component relative to any CBH typecomponents present in the cellulase composition is from preferably about1, preferably about 5, preferably about 10, preferably about 15, orpreferably about 20 weight percent to preferably about 25, preferablyabout 30, preferably about 35, preferably about 40, preferably about 45or preferably about 50 weight percent.

Furthermore, preferred ranges may be about 0.5 to about 15 weightpercent, about 0.5 to about 20 weight percent, from about 1 to about 10weight percent, from about 1 to about 15 weight percent, from about 1 toabout 20 weight percent, from about 1 to about 25 weight percent, fromabout 5 to about 20 weight percent, from about 5 to about 25 weightpercent, from about 5 to about 30 weight percent, from about 5 to about35 weight percent, from about 5 to about 40 weight percent, from about 5to about 45 weight percent, from about 5 to about 50 weight percent,from about 10 to about 20 weight percent, from about 10 to about 25weight percent, from about 10 to about 30 weight percent, from about 10to about 35 weight percent, from about 10 to about 40 weight percent,from about 10 to about 45 weight percent, from about 10 to about 50weight percent, from about 15 to about 20 weight percent, from about 15to about 25 weight percent, from about 15 to about 30 weight percent,from about 15 to about 35 weight percent, from about 15 to about 30weight percent, from about 15 to about 45 weight percent, from about 15to about 50 weight percent.

II. Target Organisms

A. Filamentous Fungi

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota. The filamentous fungi are characterized byvegetative mycelium having a cell wall composed of chitin, glucan,chitosan, mannan, and other complex polysaccharides, with vegetativegrowth by hyphal elongation and carbon catabolism that is obligatelyaerobic.

In the present invention, the filamentous fungal parent cell may be acell of a species of, but not limited to, Trichoderma, e.g., Trichodermalongibrachiatum (reesei), Trichoderma viride, Trichoderma koningii,Trichoderma harzianum; Penicillium sp.; Humicola sp., including Humicolainsolens; Chrysosporium sp., including C. lucknowense; Gliocladium sp.;Aspergillus sp.; Fusarium sp., Neurospora sp., Hypocrea sp., andEmericella sp. As used herein, the term “Trichoderma” or “Trichodermasp.” refers to any fungal strains which have previously been classifiedas Trichoderma or are currently classified as Trichoderma.

In one preferred embodiment, the filamentous fungal parent cell is anAspergillus niger, Aspergillus awamori, Aspergillus aculeatus, orAspergillus nidulans cell.

In another preferred embodiment, the filamentous fungal parent cell is aTrichoderma reesei cell.

III. Cellulases

Cellulases are known in the art as enzymes that hydrolyze cellulose(beta-1,4-glucan or beta D-glucosidic linkages) resulting in theformation of glucose, cellobiose, cellooligosaccharides, and the like.As set forth above, cellulases have been traditionally divided intothree major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanasesor cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases (EC3.2.1.21) (“BG”). (Knowles, et al., 1987; Schulein, 1988).

Certain fungi produce complete cellulase systems which includeexo-cellobiohydrolases or CBH-type cellulases, endoglucanases or EG-typecellulases and beta-glucosidases or BG-type cellulases (Schulein, 1988).However, sometimes these systems lack CBH-type cellulases and bacterialcellulases also typically include little or no CBH-type cellulases. Inaddition, it has been shown that the EG components and CBH componentssynergistically interact to more efficiently degrade cellulose. See,e.g., Wood, 1985. The different components, i.e., the variousendoglucanases and exocellobiohydrolases in a multi-component orcomplete cellulase system, generally have different properties, such asisoelectric point, molecular weight, degree of glycosylation, substratespecificity and enzymatic action patterns.

It is believed that endoglucanase-type cellulases hydrolyze internalbeta-1,4-glucosidic bonds in regions of low crystallinity of thecellulose and exo-cellobiohydrolase-type cellulases hydrolyze cellobiosefrom the reducing or non-reducing end of cellulose. It follows that theaction of endoglucanase components can greatly facilitate the action ofexo-cellobiohydrolases by creating new chain ends, which are recognizedby exo-cellobiohydrolase components. Further, beta-glucosidase-typecellulases have been shown to catalyze the hydrolysis of alkyl and/oraryl β-D-glucosides such as methyl β-D-glucoside and p-nitrophenylglucoside as well as glycosides containing only carbohydrate residues,such as cellobiose.

This yields glucose as the sole product for the microorganism andreduces or eliminates cellobiose which inhibits cellobiohydrolases andendoglucanases.

Accordingly, β-glucosidase-type cellulases are considered to be anintegral part of the cellulase system because they drive the overallreaction to glucose.

Increased expression of BG in T. reesei has been shown to improvedegradation of cellulose to glucose. See EP0562003, which is herebyincorporated by reference. In addition, β-glucosidases can catalyze thehydrolysis of a number of different substrates, and therefore they findutility in a variety of different applications. Some β-glucosidases canbe added to grapes during wine making to enhance the potential aroma ofthe finished wine product. Yet another application can be to useβ-glucosidase in fruit to enhance the aroma thereof. Alternatively,β-glucosidase can be used directly in food additives or wine processingto enhance the flavor and aroma.

Cellulases also find a number of uses in detergent compositionsincluding to enhance cleaning ability, as a softening agent and toimprove the feel of cotton fabrics (Hemmpel, 1991; Tyndall, 1992; Kumaret al., 1997). While the mechanism is not part of the invention,softening and color restoration properties of cellulase have beenattributed to the alkaline endoglucanase components in cellulasecompositions, as exemplified by U.S. Pat. Nos. 5,648,263, 5,691,178, and5,776,757, which disclose that detergent compositions containing acellulase composition enriched in a specified alkaline endoglucanasecomponent impart color restoration and improved softening to treatedgarments as compared to cellulase compositions not enriched in such acomponent. In addition, the use of such alkaline endoglucanasecomponents in detergent compositions has been shown to complement the pHrequirements of the detergent composition (e.g., by exhibiting maximalactivity at an alkaline pH of 7.5 to 10, as described in U.S. Pat. Nos.5,648,263, 5,691,178, and 5,776,757).

Cellulase compositions have also been shown to degrade cotton-containingfabrics, resulting in reduced strength loss in the fabric (U.S. Pat. No.4,822,516), contributing to reluctance to use cellulase compositions incommercial detergent applications. Cellulase compositions comprisingendoglucanase components have been suggested to exhibit reduced strengthloss for cotton-containing fabrics as compared to compositionscomprising a complete cellulase system.

Cellulases have also been shown to be useful in degradation of cellulasebiomass to ethanol (wherein the cellulase degrades cellulose to glucoseand yeast or other microbes further ferment the glucose into ethanol),in the treatment of mechanical pulp (Pere et al., 1996), for use as afeed additive (WO 91/04673) and in grain wet milling.

Most CBHs and EGs have a multidomain structure consisting of a coredomain separated from a cellulose binding domain (CBD) by a linkerpeptide (Suumakki et al., 2000). The core domain contains the activesite whereas the CBD interacts with cellulose by binding the enzyme toit (van Tilbeurgh et al., 1986; Tomme et al., 1988). The CBDs areparticularly important in the hydrolysis of crystalline cellulose. Ithas been shown that the ability of cellobiohydrolases to degradecrystalline cellulose clearly decreases when the CBD is absent (Linderand Teeri, 1997). However, the exact role and action mechanism of CBDsis still a matter of speculation. It has been suggested that the CBDenhances the enzymatic activity merely by increasing the effectiveenzyme concentration at the surface of cellulose (Stahlberg et al.,1991), and/or by loosening single cellulose chains from the cellulosesurface (Tormo et al., 1996). Most studies concerning the effects ofcellulase domains on different substrates have been carried out withcore proteins of cellobiohydrolases, as their core proteins can easilybe produced by limited proteolysis with papain (Tomme et al., 1988).Numerous cellulases have been described in the scientific literature,examples of which include: from Trichoderma reesei: Shoemaker, S. etal., Bio/Technology, 1:691-696, 1983, which discloses CBHI; Teeri, T. etal., Gene, 51:43-52, 1987, which discloses CBHII; Penttila, M. et al.,Gene, 45:253-263, 1986, which discloses EGI; Saloheimo, M. et al., Gene,63:11-22, 1988, which discloses EGII; Okada, M. et al., Appl. Environ.Microbiol., 64:555-563, 1988, which discloses EGIII; Saloheimo, M. etal., Eur. J. Biochem., 249:584-591, 1997, which discloses EGIV;Saloheimo, A. et al., Molecular Microbiology, 13:219-228, 1994, whichdiscloses EGV; Barnett, C. C., et al., Bio/Technology, 9:562-567, 1991,which discloses BGL1, and Takashima, S. et al., J. Biochem.,125:728-736, 1999, which discloses BGL2. Cellulases from species otherthan Trichoderma have also been described e.g., Ooi et al., 1990, whichdiscloses the cDNA sequence coding for endoglucanase F1-CMC produced byAspergillus aculeatus; Kawaguchi T et al., 1996, which discloses thecloning and sequencing of the cDNA encoding beta-glucosidase 1 fromAspergillus aculeatus; Sakamoto et al., 1995, which discloses the cDNAsequence encoding the endoglucanase CMCase-1 from Aspergillus kawachiiIFO 4308; Saarilahti et al., 1990 which discloses an endoglucanase fromErwinia carotovara; Spilliaert R, et al., 1994, which discloses thecloning and sequencing of bglA, coding for a thermostable beta-glucanasefrom Rhodothermus marinu; and Halldorsdottir S et al., 1998, whichdiscloses the cloning, sequencing and overexpression of a Rhodothermusmarinus gene encoding a thermostable cellulase of glycosyl hydrolasefamily 12. However, there remains a need for identification andcharacterization of novel cellulases, with improved properties, such asimproved performance under conditions of thermal stress or in thepresence of surfactants, increased specific activity, altered substratecleavage pattern, and/or high level expression in vitro.

The development of new and improved cellulase compositions that comprisevarying amounts CBH-type, EG-type and BG-type cellulases is of interestfor use: (1) in detergent compositions that exhibit enhanced cleaningability, function as a softening agent and/or improve the feel of cottonfabrics (e.g., “stone washing” or “biopolishing”); (2) in compositionsfor degrading wood pulp or other biomass into sugars (e.g., forbio-ethanol production); and/or (3) in feed compositions.

IV. Methods of Identifying Novel Sequences

Open reading frames (ORFs) are analyzed following full or partialsequencing of the T. reesei genome or of clones of cDNA librariesderived from T. reesei mRNA and are further analyzed using sequenceanalysis software, and by determining homology to known sequences indatabases (public/private).

V. egl6 Nucleic Acids and EGVI Polypeptides.

A. egl6 Nucleic Acids

The nucleic acid molecules of the present invention include the nativecoding sequence for egl6. In one embodiment the sequence is the cDNAsequence for egl6 presented herein as SEQ. ID. NO:1 or SEQ. ID. NO:4,and homologues thereof in other species, naturally occurring allelic andsplice variants, nucleic acid fragments, and biologically active(functional) derivatives thereof, such as, amino acid sequence variantsof the native molecule and sequences which encode fusion proteins. Thesequences are collectively referred to herein as “EGVI-encoding nucleicacid sequences”.

A Basic BLASTN search (http://www.ncbi.nlm.nih.gov/BLAST) of thenon-redundant nucleic acid sequence database was conducted on Sep. 12,2001, with the egl6 gene sequence presented in FIG. 1 (SEQ ID NO:1),indicated no sequences producing significant alignments (i.e. with an Evalue of less than 10.

Part of the egl6 sequence presented in FIG. 1 (SEQ ID NO:1) is identicalto parts of the sequences of Trichoderma reesei ESTs disclosed as SEQ IDNO:7511 and SEQ ID NO:7641 and annotated as a endoglucanases in patentapplication WO 0056762.

An egl6 nucleic acid sequence of this invention may be a DNA or RNAsequence, derived from genomic DNA, cDNA, mRNA, or may be synthesized inwhole or in part. The DNA may be double-stranded or single-stranded andif single-stranded may be the coding strand or the non-coding(antisense, complementary) strand. The nucleic acid sequence may becloned, for example, by isolating genomic DNA from an appropriatesource, and amplifying and cloning the sequence of interest using apolymerase chain reaction (PCR). Alternatively, nucleic acid sequencemay be synthesized, either completely or in part, especially where it isdesirable to provide host-preferred sequences for optimal expression.Thus, all or a portion of the desired structural gene (that portion ofthe gene which encodes a polypeptide or protein) may be synthesizedusing codons preferred by a selected host.

Due to the inherent degeneracy of the genetic code, nucleic acidsequences other than the native form which encode substantially the sameor a functionally equivalent amino acid sequence may be used to cloneand/or express EGVI-encoding nucleic acid sequences. Thus, for a givenEGVI-encoding nucleic add sequence, it is appreciated that as a resultof the degeneracy of the genetic code, a number of coding sequences canbe produced that encode a protein having the same amino acid sequence.For example, the triplet CGT encodes the amino add arginine. Arginine isalternatively encoded by CGA, CGC, CGG, AGA, and AGG. Therefore it isappreciated that such substitutions in the coding region fall within thenucleic add sequence variants covered by the present invention. Any andall of these sequence variants can be utilized in the same way asdescribed herein for the native form of an EGVI-encoding nucleic acidsequence.

A “variant” EGVI-encoding nucleic acid sequence may encode a “variant”EGVI amino acid sequence which is altered by one or more amino acidsfrom the native polypeptide sequence or may be truncated by removal ofone or more amino acids from either end of the polypeptide sequence,both of which are included within the scope of the invention. Similarly,the term “modified form of”, relative to EGVI, means a derivative orvariant form of the native EGVI protein-encoding nucleic acid sequenceor the native EGVI amino acid sequence.

Similarly, the polynucleotides for use in practicing the inventioninclude sequences, which encode native EGVI proteins and splice variantsthereof, sequences complementary to the native protein coding sequence,and novel fragments of EGVI encoding polynucleotides. An EGVI encodingnucleic acid sequence may contain one or more intron sequences if it isa genomic DNA sequence.

In one general embodiment, an EGVI-encoding nucleotide sequence has atleast 70%, preferably 80%, 85%, 90%, 95%, 98%, or more sequence identityto the egl6 coding sequence presented herein as SEQ ID NO:1.

In another embodiment, an EGVI-encoding nucleotide sequence willhybridize under moderate to high stringency conditions to a nucleotidesequence that encodes an EGVI protein. In a related embodiment, anEGVI-encoding nucleotide sequence will hybridize under moderate to highstringency conditions to the nucleotide sequence presented as SEQ IDNO:1.

It is appreciated that some nucleic acid sequence variants that encodeEGVI may or may not selectively hybridize to the parent sequence. By wayof example, in situations where the coding sequence has been optimizedbased on the degeneracy of the genetic code, a variant coding sequencemay be produced that encodes an EGVI protein, but does not hybridize toa native EGVI-encoding nucleic acid sequence under moderate to highstringency conditions. This would occur, for example, when the sequencevariant includes a different codon for each of the amino adds encoded bythe parent nucleotide.

As will be further understood by those of skill in the art, in somecases it may be advantageous to produce nucleotide sequences possessingnon-naturally occurring codons e.g., inosine or other non-naturallyoccurring nucleotide analog. Codons preferred by a particular eukaryotichost can be selected, for example, to increase the rate of EGVI proteinexpression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced fromthe naturally occurring sequence. Hence, a native EGVI-encodingnucleotide sequence may be engineered in order to alter the codingsequence for a variety of reasons, including but not limited to,alterations which modify the cloning, processing and/or expression ofthe EGVI protein by a cell.

Particularly preferred are nucleic acid substitutions, additions, anddeletions that are silent such that they do not alter the properties oractivities of the native polynucleotide or polypeptide.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, and PCRmutagenesis. Site-directed mutagenesis (Carter et al., 1986; Zoller etal., 1987), cassette mutagenesis (Wells et al., 1985), restrictionselection mutagenesis (Wells et al., 1986) or other known techniques canbe performed on the cloned DNA to produce the EGVI polypeptide-encodingvariant DNA.

However, in some cases it may be advantageous to express variants ofegl6 which lack the properties or activities of the native egl6polynucleotide or EGVI polypeptide. In such cases, mutant or modifiedforms of the native EGVI-encoding nucleic acid sequence may be generatedusing techniques routinely employed by those of skill in the art.

B. EGVI Polypeptides

In one preferred embodiment, the invention provides an EGVI polypeptide,having a native mature or full-length EGVI polypeptide sequencecomprising the sequence presented in FIG. 2 (SEQ ID NO:2). An EGVIpolypeptide of the invention can be the mature EGVI polypeptide, part ofa fusion protein or a fragment or variant of the EGVI polypeptidesequence presented in FIG. 2 (SEQ ID NO:2).

Ordinarily, an EGVI polypeptide of the invention has at least 80%identity to an EGVI amino acid sequence over its entire length. Morepreferable are EGVI polypeptide sequences that comprise a region havingat least 80, 85, 90, 95, 98% or more sequence identity to the EGVIpolypeptide sequence of FIG. 2 (SEQ ID NO:2), using a sequence alignmentprogram, as detailed herein.

Typically, a “modified form of” a native EGVI protein or a “variant”EGVI protein has a derivative sequence containing at least one aminoacid substitution, addition, deletion or insertion, respectively.

It is well known in the art that certain amino acid substitutions may bemade in protein sequences without affecting the function of the protein.Generally, conservative amino acid substitutions or substitutions ofsimilar amino acids are tolerated without affecting protein function.Similar amino acids can be those that are similar in size and/or chargeproperties, for example, aspartate and glutamate, and isoleucine andvaline, are both pairs of similar amino acids. Similarity between aminoacid pairs has been assessed in the art in a number of ways. Forexample, Dayhoff et al. (1978), which is incorporated by referenceherein provides frequency tables for amino acid substitutions which canbe employed as a measure of amino acid similarity. Dayhoff et al.'sfrequency tables are based on comparisons of amino acid sequences forproteins having the same function from a variety of evolutionarilydifferent sources.

Fragments and variants of the EGVI polypeptide sequence of FIG. 2 (SEQID NO:2), are considered to be a part of the invention. A fragment is avariant polypeptide which has an amino acid sequence that is entirelythe same as part but not all of the amino acid sequence of thepreviously described polypeptides. The fragments can be “free-standing”or comprised within a larger polypeptide of which the fragment forms apart or a region, most preferably as a single continuous region.Preferred fragments are biologically active fragments which are thosefragments that mediate activities of the polypeptides of the invention,including those with similar activity or improved activity or with adecreased activity. Also included are those fragments that are antigenicor immunogenic in an animal, particularly a human. In this aspect, theinvention includes (i) fragments of EGVI, preferably at least about20-100 amino acids in length, more preferably about 100-200 amino acidsin length, and (ii) a pharmaceutical composition comprising EGVI. Invarious embodiments, the fragment corresponds to the N-terminal domainof EGVI or the C-terminal domain of EGVI.

EGVI polypeptides of the invention also include polypeptides that varyfrom the EGVI polypeptide sequence of FIG. 2 (SEQ ID NO:2). Thesevariants may be substitutional, insertional or deletional variants. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics as further described below.

A “substitution” results from the replacement of one or more nucleotidesor amino acids by different nucleotides or amino acids, respectively.

An “insertion” or “addition” is that change in a nucleotide or aminoacid sequence which has resulted in the addition of one or morenucleotides or amino acid residues, respectively, as compared to thenaturally occurring sequence.

A “deletion” is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, are absent.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances.

Amino acid substitutions can be the result of replacing one amino acidwith another amino acid having similar structural and/or chemicalproperties, such as the replacement of an isoleucine with a valine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of 1 to 5 amino acids.

Substitutions are generally made in accordance with known “conservativesubstitutions”. A “conservative substitution” refers to the substitutionof an amino acid in one class by an amino acid in the same class, wherea class is defined by common physicochemical amino acid side chainproperties and high substitution frequencies in homologous proteinsfound in nature (as determined, e.g., by a standard Dayhoff frequencyexchange matrix or BLOSUM matrix). (See generally, Doolittle, R. F.,1986.)

A “non-conservative substitution” refers to the substitution of an aminoacid in one class with an amino acid from another class.

EGVI polypeptide variants typically exhibit the same qualitativebiological activity as the naturally-occurring analogue, althoughvariants also are selected to modify the characteristics of the EGVIpolypeptide, as needed. For example, glycosylation sites, and moreparticularly one or more O-linked or N-linked glycosylation sites may bealtered or removed. Those skilled in the art will appreciate that aminoacid changes may alter post-translational processes of the EGVIpolypeptide, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics or secretioncharacteristics or other cellular localization characteristics.

Also included within the definition of EGVI polypeptides are otherrelated EGVI polypeptides. Thus, probe or degenerate polymerase chainreaction (PCR) primer sequences may be used to find other relatedpolypeptides. Useful probe or primer sequences may be designed to: allor part of the EGVI polypeptide sequence, or sequences outside thecoding region. As is generally known in the art, preferred PCR primersare from about 15 to about 35 nucleotides in length, with from about 20to about 30 being preferred, and may contain inosine as needed. Theconditions for the PCR reaction are generally known in the art.

Covalent modifications of EGVI polypeptides are also included within thescope of this invention. For example, the invention provides EGVIpolypeptides that are a mature protein and may comprise additional aminoor carboxyl-terminal amino acids, or amino acids within the maturepolypeptide (for example, when the mature form of the protein has morethan one polypeptide chain). Such sequences can, for example, play arole in the processing of the protein from a precursor to a mature form,allow protein transport, shorten or lengthen protein half-life, orfacilitate manipulation of the protein in assays or production.

Also contemplated are modifications directed to alteration of an activesite, alteration of the pH optima, temperature optima, and/or substrateaffinity of the EGVI enzyme.

FIG. 2 shows the predicted amino acid sequence (SEQ ID NO:2) of anexemplary EGVI polypeptide based on the nucleotide sequence provided inFIG. 1. The predicted molecular weight of the encoded EGVI polypeptideis 87.1 kDa. A predicted signal peptide of 19 amino acids precedes themature amino terminus of EGVI as provided in the figure suggesting thatthe EGVI polypeptide is secreted (Nielsen, H., Engelbrecht, J., Brunak,S., von Heijne, G., Protein Engineering, 10:1-6, 1997). The last 35amino acids of the sequence (SEQ ID NO:2) has up to 65% identity withthe cellulose binding domains present on many fungal secreted cellulasesand hemicellulases (Tomme, P., Warren, R. A., Miller, R. C., Jr.,Kilbum, D. G. & Gilkes, N. R. (1995) in Enzymatic Degradation ofInsoluble Polysaccharides (Saddler, J. N. & Penner, M., eds.),Cellulose-binding domains: classification and properties. pp. 142-163,American Chemical Society, Washington). The amino acids fromapproximately residue number 744 to approximately residue number 801have the characteristics of a serine- and threonine-rich linker regionwhich is generally found between the cellulose binding domain and thecatalytic region of fungal enzymes having a cellulose binding domain.

A Basic BLASTP search (http://www.ncbi.nlm.nih.gov/BLAST) of thenon-redundant protein database, conducted on Sep. 12, 2001 with the EGVIamino acid sequence indicated 51% identity with GenBank Accession NumberAB015511 (avicellase III of Aspergillus aculeatus), 49% sequenceidentity to GenBank Accession Number AJ292929 (CEL6 protein of Agaricusbisponis), 39% sequence identity to GenBank Accession Number AE007608(probable secreted sialidase of Clostridium acetabutylicum), and 40%sequence identity to GenBank Accession Number AL031515 (probablesecreted cellulase of Streptomyces coelicolor). These sequencesimilarities indicate that EGVI is a member of glycosyl hydrolase family74 (Henrissat, B. and Bairoch, A. (1993) Biochem. J. 293:781-788).

C. Anti-EGVI Antibodies.

The present invention further provides anti-EGVI antibodies. Theantibodies may be polyclonal, monoclonal, humanized, bispecific orheteroconjugate antibodies.

Methods of preparing polyclonal antibodies are known to the skilledartisan. The immunizing agent may be an EGVI polypeptide or a fusionprotein thereof. It may be useful to conjugate the antigen to a proteinknown to be immunogenic in the mammal being immunized. The immunizationprotocol may be determined by one skilled in the art based on standardprotocols or routine experimentation.

Alternatively, the anti-EGVI antibodies may be monoclonal antibodies.Monoclonal antibodies may be produced by cells immunized in an animal orusing recombinant DNA methods. (See, e.g., Kohler et al., 1975; U.S.Pat. No. 4,816,567).

An anti-EGVI antibody of the invention may further comprise a humanizedor human antibody. The term “humanized antibody” refers to humanizedforms of non-human (e.g., murine) antibodies that are chimericantibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab,Fab′, F(ab′)₂ or other antigen-binding partial sequences of antibodies)which contain some portion of the sequence derived from non-humanantibody. Methods for humanizing non-human antibodies are well known inthe art, as further detailed in Jones et al., 1986; Riechmann et al.,1988; and Verhoeyen et al., 1988. Methods for producing human antibodiesare also known in the art. See, e.g., Jakobovits, A, et al., 1995 andJakobovits, A, 1995.

VI. Expression Of Recombinant EGVI

The methods of the invention rely on the use cells to express EGVI, withno particular method of EGVI expression required.

The invention provides host cells which have been transduced,transformed or transfected with an expression vector comprising anEGVI-encoding nucleic acid sequence. The culture conditions, such astemperature, pH and the like, are those previously used for the parentalhost cell prior to transduction, transformation or transfection and willbe apparent to those skilled in the art.

In one approach, a filamentous fungal cell or yeast cell is transfectedwith an expression vector having a promoter or biologically activepromoter fragment or one or more (e.g., a series) of enhancers whichfunctions in the host cell line, operably linked to a DNA segmentencoding EGVI, such that EGVI is expressed in the cell line.

A. Nucleic Acid Constructs/Expression Vectors.

Natural or synthetic polynucleotide fragments encoding EGVI(“EGVI-encoding nucleic acid sequences”) may be incorporated intoheterologous nucleic acid constructs or vectors, capable of introductioninto, and replication in, a filamentous fungal or yeast cell. Thevectors and methods disclosed herein are suitable for use in host cellsfor the expression of EGVI. Any vector may be used as long as it isreplicable and viable in the cells into which it is introduced. Largenumbers of suitable vectors and promoters are known to those of skill inthe art, and are commercially available. Cloning and expression vectorsare also described in Sambrook et al., 1989, Ausubel F M et al., 1989,and Strathern et al., 1981, each of which is expressly incorporated byreference herein. Appropriate expression vectors for fungi are describedin van den Hondel, C. A. M. J. J. et al. (1991) In: Bennett, J. W. andLasure, L. L. (eds.) More Gene Manipulations in Fungi. Academic Press,pp. 396-428. The appropriate DNA sequence may be inserted into a plasmidor vector (collectively referred to herein as “vectors”) by a variety ofprocedures. In general, the DNA sequence is inserted into an appropriaterestriction endonuclease site(s) by standard procedures. Such proceduresand related sub-cloning procedures are deemed to be within the scope ofknowledge of those skilled in the art.

Recombinant filamentous fungi comprising the coding sequence for EGVImay be produced by introducing a heterologous nucleic acid constructcomprising the EGVI coding sequence into the cells of a selected strainof the filamentous fungi.

Once the desired form of an egl6 nucleic acid sequence, homologue,variant or fragment thereof, is obtained, it may be modified in avariety of ways. Where the sequence involves non-coding flankingregions, the flanking regions may be subjected to resection,mutagenesis, etc. Thus, transitions, transversions, deletions, andinsertions may be performed on the naturally occurring sequence.

A selected egl6 coding sequence may be inserted into a suitable vectoraccording to well-known recombinant techniques and used to transformfilamentous fungi capable of EGVI expression. Due to the inherentdegeneracy of the genetic code, other nucleic acid sequences whichencode substantially the same or a functionally equivalent amino acidsequence may be used to clone and express EGVI. Therefore it isappreciated that such substitutions in the coding region fall within thesequence variants covered by the present invention. Any and all of thesesequence variants can be utilized in the same way as described hereinfor a parent EGVI-encoding nucleic acid sequence.

The present invention also includes recombinant nucleic acid constructscomprising one or more of the EGVI-encoding nucleic acid sequences asdescribed above. The constructs comprise a vector, such as a plasmid orviral vector, into which a sequence of the invention has been inserted,in a forward or reverse orientation.

Heterologous nucleic acid constructs may include the coding sequence foregl6, or a variant, fragment or splice variant thereof: (i) inisolation; (ii) in combination with additional coding sequences; such asfusion protein or signal peptide coding sequences, where the egl6 codingsequence is the dominant coding sequence; (iii) in combination withnon-coding sequences, such as introns and control elements, such aspromoter and terminator elements or 5′ and/or 3′ untranslated regions,effective for expression of the coding sequence in a suitable host;and/or (iv) in a vector or host environment in which the egl6 codingsequence is a heterologous gene.

In one aspect of the present invention, a heterologous nucleic acidconstruct is employed to transfer an EGVI-encoding nucleic acid sequenceinto a cell in vitro, with established filamentous fungal and yeastlines preferred. For long-term, high-yield production of EGVI, stableexpression is preferred. It follows that any method effective togenerate stable transformants may be used in practicing the invention.

Appropriate vectors are typically equipped with a selectablemarker-encoding nucleic acid sequence, insertion sites, and suitablecontrol elements, such as promoter and termination sequences. The vectormay comprise regulatory sequences, including, for example, non-codingsequences, such as introns and control elements, i.e., promoter andterminator elements or 5′ and/or 3′ untranslated regions, effective forexpression of the coding sequence in host cells (and/or in a vector orhost cell environment in which a modified soluble protein antigen codingsequence is not normally expressed), operably linked to the codingsequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, many of which are commercially availableand/or are described in Sambrook, et al., (supra).

Exemplary promoters include both constitutive promoters and induciblepromoters, examples of which include a CMV promoter, an SV40 earlypromoter, an RSV promoter, an EF-1α promoter, a promoter containing thetet responsive element (TRE) in the tet-on or tet-off system asdescribed (ClonTech and BASF), the beta actin promoter and themetallothionine promoter that can upregulated by addition of certainmetal salts. A promoter sequence is a DNA sequence which is recognizedby the particular filamentous fungus for expression purposes. It isoperably linked to DNA sequence encoding an EGVI polypeptide. Suchlinkage comprises positioning of the promoter with respect to theinitiation codon of the DNA sequence encoding the EGVI polypeptide inthe disclosed expression vectors. The promoter sequence containstranscription and translation control sequence which mediate theexpression of the EGVI polypeptide. Examples include the promoters fromthe Aspergillus niger, A awamori or A. oryzae glucoamylase,alpha-amylase, or alpha-glucosidase encoding genes; the A. nidulans gpdAor trpC Genes; the Neurospora crassa cbh1 or trp1 genes; the A. niger orRhizomucor miehei aspartic proteinase encoding genes; the T. reeseicbh1, cbh2, egl1, egl2, or other cellulase encoding genes.

The choice of the proper selectable marker will depend on the host cell,and appropriate markers for different hosts are well known in the art.Typical selectable marker genes include argB from A. nidulans or T.reesei, amdS from A. nidulans, pyr4 from Neurospora crassa or T. reesei,pyrG from Aspergillus niger or A. nidulans. Additional exemplaryselectable markers include, but are not limited to trpc, trp1, oliC31,niaD or leu2, which are included in heterologous nucleic acid constructsused to transform a mutant strain such as trp-, pyr-, leu- and the like.

Such selectable markers confer to transformants the ability to utilize ametabolite that is usually not metabolized by the filamentous fungi. Forexample, the amdS gene from T. reesei which encodes the enzymeacetamidase that allows transformant cells to grow on acetamide as anitrogen source. The selectable marker (e.g. pyrG) may restore theability of an auxotrophic mutant strain to grow on a selective minimalmedium or the selectable marker (e.g. olic31) may confer totransformants the ability to grow in the presence of an inhibitory drugor antibiotic.

The selectable marker coding sequence is cloned into any suitableplasmid using methods generally employed in the art. Exemplary plasmidsinclude pUC18, pBR322, and pUC100.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Sambrook et al., 1989; Freshney, 1987; Ausubel, et al., 1993; andColigan et al., 1991. All patents, patent applications, articles andpublications mentioned herein, are hereby expressly incorporated hereinby reference.

B. Host Cells and Culture Conditions For Enhanced EGVI Production

(i) Filamentous Fungi

Thus, the present invention provides filamentous fungi comprising cellswhich have been modified, selected and cultured in a manner effective toresult in enhanced EGVI production or expression relative to thecorresponding non-transformed parental fungi.

Examples of species of parental filamentous fungi that may be treatedand/or modified for enhanced EGVI expression include, but are notlimited to Trichoderma, e.g., Trichoderma reesei, Trichodermalongibrachiatum, Trichoderma viride, Trichoderma koningii; Penicilliumsp., Humicola sp., including Humicola insolens; Aspergillus sp.,Chrysosporium sp., Fusanum sp., Hypocrea sp., and Emericella sp.

EGVI expressing cells are cultured under conditions typically employedto culture the parental fungal line. Generally, cells are cultured in astandard medium containing physiological salts and nutrients, such asdescribed in Pourquie, J. et al., Biochemistry and Genetics of CelluloseDegradation, eds. Aubert, J. P. et al., Academic Press, pp. 71-86, 1988and Ilmen, M. et al., Appl. Environ. Microbiol. 63:1298-1306, 1997.Culture conditions are also standard, e.g., cultures are incubated at28° C. in shaker cultures or fermenters until desired levels of EGVIexpression are achieved.

Preferred culture conditions for a given filamentous fungus may be foundin the scientific literature and/or from the source of the fungi such asthe American Type Culture Collection (ATCC; “http://www.atcc.org/”).After fungal growth has been established, the cells are exposed toconditions effective to cause or permit the over expression of EGVI.

In cases where an EGVI coding sequence is under the control of aninducible promoter, the inducing agent, e.g., a sugar, metal salt orantibiotics, is added to the medium at a concentration effective toinduce high-level EGVI expression.

(ii) Yeast

The present invention also contemplates the use of yeast as a host cellfor EGVI production. Several other genes encoding hydrolytic enzymeshave been expressed in various strains of the yeast S. cerevisiae. Theseinclude sequences encoding for two endoglucanases (Penttila et al.,1987), two cellobiohydrolases (Penttila et al., 1988) and onebeta-glucosidase from Trichoderma reesei (Cummings and Fowler, 1996), axylanase from Aureobasidlium pullulans (Li and Ljungdahl, 1996), analpha-amylase from wheat (Rothstein et al., 1987), etc. In addition, acellulase gene cassette encoding the Butyrivibrio fibrisolvensendo-[beta]-1,4-glucanase (END1), Phanerochaete chrysosporiumcellobiohydrolase (CBH1), the Ruminococcus flavefaciens cellodextrinase(CEL1) and the Endomyces fibrilizer cellobiase (Bgl1) was successfullyexpressed in a laboratory strain of S. cerevisiae (Van Rensburg et al.,1998).

C. Introduction of an EGVI-Encoding Nucleic Acid Sequence into HostCells.

The invention further provides cells and cell compositions which havebeen genetically modified to comprise an exogenously providedEGVI-encoding nucleic acid sequence. A parental cell or cell line may begenetically modified (i.e., transduced, transformed or transfected) witha cloning vector or an expression vector. The vector may be, forexample, in the form of a plasmid, a viral particle, a phage, etc, asfurther described above.

Various methods may be employed for delivering an expression vector intocells in vitro. After a suitable vector is constructed, it is used totransform strains of fungi or yeast. General methods of introducingnucleic acids into cells for expression of heterologous nucleic acidsequences are known to the ordinarily skilled artisan. Such methodsinclude, but not limited to, electroporation; nuclear microinjection ordirect microinjection into single cells; bacterial protoplast fusionwith intact cells; use of polycations, e.g., polybrene or polyomithine;membrane fusion with liposomes, lipofectamine or lipofection-mediatedtransfection; high velocity bombardment with DNA-coatedmicroprojectiles; incubation with calcium phosphate-DNA precipitate;DEAE-Dextran mediated transfection; infection with modified viralnucleic acids; and the like.

Preferred methods for introducing a heterologous nucleic acid construct(expression vector) into filamentous fungi (e.g., T. reesei) include,but are not limited to the use of a particle or gene gun,permeabilization of filamentous fungi cells walls prior to thetransformation process (e.g., by use of high concentrations of alkali,e.g., 0.05 M to 0.4 M CaCl₂ or lithium acetate), protoplast fusion oragrobacterium mediated transformation. An exemplary method fortransformation of filamentous fungi by treatment of protoplasts orspheroplasts with polyethylene glycol and CaCl₂ is described inCampbell, E. I. et al., Curr. Genet. 16:53-56, 1989 and Penttila, M. etal., Gene, 63:11-22, 1988.

In addition, heterologous nucleic acid constructs comprising anEGVI-encoding nucleic acid sequence can be transcribed in vitro, and theresulting RNA introduced into the host cell by well-known methods, e.g.,by injection.

Following introduction of a heterologous nucleic acid constructcomprising the coding sequence for egl6, the genetically modified cellscan be cultured in conventional nutrient media modified as appropriatefor activating promoters, selecting transformants or amplifyingexpression of an EGVI-encoding nucleic acid sequence. The cultureconditions, such as temperature, pH and the like, are those previouslyused for the host cell selected for expression, and will be apparent tothose skilled in the art.

The progeny of cells into which such heterologous nucleic acidconstructs have been introduced are generally considered to comprise theEGVI-encoding nucleic acid sequence found in the heterologous nucleicacid construct.

The invention further includes novel and useful transformants offilamentous fungi such as Trichoderma reesei for use in producing fungalcellulase compositions. The invention includes transformants offilamentous fungi especially fungi comprising the egl6 coding sequence,comprising a modified form of the egl6 coding sequence or deletion ofthe egl6 coding sequence.

Stable transformants of filamentous fungi can generally be distinguishedfrom unstable transformants by their faster growth rate and theformation of circular colonies with a smooth rather than ragged outlineon solid culture medium. Additionally, in some cases, a further test ofstability can be made by growing the transformants on solidnon-selective medium, harvesting the spores from this culture medium anddetermining the percentage of these spores which will subsequentlygerminate and grow on selective medium.

VII. Analysis For EGVI Nucleic Acid Coding Sequences and/or ProteinExpression.

In order to evaluate the expression of EGVI by a cell line that has beentransformed with an EGVI-encoding nucleic acid construct, assays can becarried out at the protein level, the RNA level or by use of functionalbioassays particular to endoglucanase activity and/or production.

In one exemplary application of the egl6 nucleic acid and proteinsequences described herein, a genetically modified strain of filamentousfungi, e.g., Trichoderma reesei, is engineered to produce an increasedamount of EGVI. Such genetically modified filamentous fungi would beuseful to produce a cellulase product with greater increasedcellulolytic capacity. In one approach, this is accomplished byintroducing the coding sequence for egl6 into a suitable host, e.g., afilamentous fungi such as Trichoderma reesei.

Accordingly, the invention includes methods for expressing EGVI in afilamentous fungus or other suitable host by introducing an expressionvector containing the DNA sequence encoding EGVI into cells of thefilamentous fungus or other suitable host.

In another aspect, the invention includes methods for modifying theexpression of EGVI in a filamentous fungus or other suitable host. Suchmodification includes a decrease or elimination in expression, orexpression of an altered form of EGVI. An altered form of EGVI may havean altered amino acid sequence or an altered nucleic acid sequence.

In general, assays employed to analyze the expression of EGVI include,Northern blotting, dot blotting (DNA or RNA analysis), RT-PCR (reversetranscriptase polymerase chain reaction), or in situ hybridization,using an appropriately labeled probe (based on the nucleic acid codingsequence) and conventional Southern blotting and autoradiography.

In addition, the production and/or expression of EGVI may be measured ina sample directly, for example, by assays for endoglucanase activity,expression and/or production. Such assays are described, for example, inShoemaker, S. P. and Brown, R. D. Jr. (Biochim. Biophys. Acta, 1978,523:133-146; Schulein (1988) and U.S. Pat. Nos. 5,246,853 and 5,475,101each of which is expressly incorporated by reference herein. The abilityof EGVI to hydrolyze isolated soluble and insoluble substrates can bemeasured using assays described in Suumakki et al. (2000) and Ortega etal. (2001). Substrates useful for assaying cellobiohydrolase,endoglucanase or β-glucosidase activities include crystalline cellulose,filter paper, phosphoric acid swollen cellulose, hydroxyethyl cellulose,carboxymethyl cellulose, cellooligosaccharides, methylumbelliferyllactoside, methylumbelliferyl cellobioside, orthonitrophenyl lactoside,paranitrophenyl lactoside, orthonitrophenyl cellobioside,paranitrophenyl cellobioside, orthonitrophenyl glucoside,paranitrophenyl glucoside, methylumbelliferyl glycoside

In addition, protein expression, may be evaluated by immunologicalmethods, such as immunohistochemical staining of cells, tissue sectionsor immunoassay of tissue culture medium, e.g., by Western blot or ELISA.Such immunoassays can be used to qualitatively and quantitativelyevaluate expression of EGVI. The details of such methods are known tothose of skill in the art and many reagents for practicing such methodsare commercially available.

A purified form of EGVI may be used to produce either monoclonal orpolyclonal antibodies specific to the expressed protein for use invarious immunoassays. (See, e.g., Hu et al., 1991). Exemplary assaysinclude ELISA, competitive immunoassays, radioimmunoassays, Westernblot, indirect immunofluorescent assays and the like. In general,commercially available antibodies and/or kits may be used for thequantitative immunoassay of the expression level of endoglucanaseproteins.

VIII. Isolation and Purification of Recombinant EGVI Protein.

In general, an EGVI protein produced in cell culture is secreted intothe medium and may be purified or isolated, e.g., by removing unwantedcomponents from the cell culture medium. However, in some cases, an EGVIprotein may be produced in a cellular form necessitating recovery from acell lysate. In such cases the EGVI protein is purified from the cellsin which it was produced using techniques routinely employed by those ofskill in the art. Examples include, but are not limited to, affinitychromatography (Tilbeurgh et al., 1984), ion-exchange chromatographicmethods (Goyal et al., 1991; Fliess et al., 1983; Bhikhabhai et al.,1984; Ellouz et al., 1987), including ion-exchange using materials withhigh resolution power (Medve et al., 1998), hydrophobic interactionchromatography (Tomaz and Queiroz, 1999), and two-phase partitioning(Brumbauer, et al., 1999).

Typically, the EGVI protein is fractionated to segregate proteins havingselected properties, such as binding affinity to particular bindingagents, e.g., antibodies or receptors; or which have a selectedmolecular weight range, or range of isoelectric points.

Once expression of a given EGVI protein is achieved, the EGVI proteinthereby produced is purified from the cells or cell culture. Exemplaryprocedures suitable for such purification include the following:antibody-affinity column chromatography, ion exchange chromatography;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; and gel filtration using, e.g., SephadexG-75. Various methods of protein purification may be employed and suchmethods are known in the art and described e.g. in Deutscher, 1990;Scopes, 1982. The purification step(s) selected will depend, e.g., onthe nature of the production process used and the particular proteinproduced.

IX. Utility of egl6 and EGVI

It can be appreciated that the egl6 nucleotide, the EGVI protein andcompositions comprising EGVI protein activity find utility in a widevariety applications, some of which are described below.

New and improved cellulase compositions that comprise varying amountsCBH-type, EG-type and BG-type cellulases find utility in detergentcompositions that exhibit enhanced cleaning ability, function as asoftening agent and/or improve the feel of cotton fabrics (e.g., “stonewashing” or “biopolishing”), in compositions for degrading wood pulpinto sugars (e.g., for bio-ethanol production), and/or in feedcompositions. The isolation and characterization of cellulase of eachtype provides the ability to control the aspects of such compositions.

In one preferred approach, the cellulase of the invention finds utilityin detergent compositions or in the treatment of fabrics to improve thefeel and appearance.

Since the rate of hydrolysis of cellulosic products may be increased byusing a transformant having at least one additional copy of the egl6gene inserted into the genome, products that contain cellulose orheteroglycans can be degraded at a faster rate and to a greater extent.Products made from cellulose such as paper, cotton, cellulosic diapersand the like can be degraded more efficiently in a landfill. Thus, thefermentation product obtainable from the transformants or thetransformants alone may be used in compositions to help degrade byliquefaction a variety of cellulose products that add to the overcrowdedlandfills.

Separate saccharification and fermentation is a process wherebycellulose present in biomass, e.g., corn stover, is converted to glucoseand subsequently yeast strains convert glucose into ethanol.Simultaneous saccharification and fermentation is a process wherebycellulose present in biomass, e.g., corn stover, is converted to glucoseand, at the same time and in the same reactor, yeast strains convertglucose into ethanol. Thus, in another preferred approach, theglucosidase type cellulase of the invention finds utility in thedegradation of biomass to ethanol. Ethanol production from readilyavailable sources of cellulose provides a stable, renewable fuel source.

Cellulose-based feedstocks are comprised of agricultural wastes, grassesand woods and other low-value biomass such as municipal waste (e.g.,recycled paper, yard clippings, etc.). Ethanol may be produced from thefermentation of any of these cellulosic feedstocks. However, thecellulose must first be converted to sugars before there can beconversion to ethanol.

A large variety of feedstocks may be used with the inventiveendoglucanase and the one selected for use may depend on the regionwhere the conversion is being done. For example, in the MidwesternUnited States agricultural wastes such as wheat straw, corn stover andbagasse may predominate while in California rice straw may predominate.However, it should be understood that any available cellulosic biomassmay be used in any region.

A cellulase composition containing an enhanced amount of endoglucanasefinds utility in ethanol production. Ethanol from this process can befurther used as an octane enhancer or directly as a fuel in lieu ofgasoline which is advantageous because ethanol as a fuel source is moreenvironmentally friendly than petroleum derived products. It is knownthat the use of ethanol will improve air quality and possibly reducelocal ozone levels and smog. Moreover, utilization of ethanol in lieu ofgasoline can be of strategic importance in buffering the impact ofsudden shifts in non-renewable energy and petrochemical supplies.

Ethanol can be produced via saccharification and fermentation processesfrom cellulosic biomass such as trees, herbaceous plants, municipalsolid waste and agricultural and forestry residues. However, the ratioof individual cellulase enzymes within a naturally occuring cellulasemixture produced by a microbe may not be the most efficient for rapidconversion of cellulose in biomass to glucose. It is known thatendoglucanases act to produce new cellulose chain ends which themselvesare substrates for the action of cellobiohydrolases and thereby improvethe efficiency of hydrolysis of the entire cellulase system. Therefore,the use of increased or optimized endoglucanase activity would greatlyenhance the production of ethanol.

Thus, the inventive endoglucanase finds use in the hydrolysis ofcellulose to its sugar components. In one embodiment, the endoglucanaseis added to the biomass prior to the addition of a fermentativeorganism. In a second embodiment, the endoglucanase is added to thebiomass at the same time as a fermentative organism. Optionally, theremay be other cellulase components present in either embodiment.

In another embodiment the cellulosic feedstock may be pretreated.Pretreatment may be by elevated temperature and the addition of eitherof dilute acid, concentrated acid or dilute alkali solution. Thepretreatment solution is added for a time sufficient to at leastpartially hydrolyze the hemicellulose components and then neutralized.

In an alternative approach, a cellulase composition, which is deficientin or free of endoglucanase, is preferred. The deletion of theendoglucanase gene of this invention would be particularly useful inpreparing cellulase compositions for use in detergents. Additionally,such compositions are useful for the production ofcellooligosaccharides. The deletion of the egl6 gene from T. reeseistrains would be particularly useful in preparing cellulase compositionsfor use in the detergents and in isolating cellooligosaccharides. Thecellulase enzymes have been used in a variety of detergent compositionsto enzymatically clean clothes. However, it is known in this art thatuse of cellulase enzymes can impart degradation of the cellulose fibersin clothes. One possibility to decrease the degradation effect is toproduce a detergent that does not contain endoglucanase. Thus, thedeletion of this protein would effect the cellulase system to inhibitthe other components via accumulation of cellobiose. The modifiedmicroorganisms of this invention are particularly suitable for preparingsuch compositions because the egl6 gene can be deleted leaving theremaining CBH and EG components resulting in improved cleaning andsoftening benefits in the composition without degradative effects.

The detergent compositions of this invention may employ besides thecellulase composition (irrespective of the endoglucanase content, i.e.,endoglucanase-free, substantially endoglucanase-free, or endoglucanaseenhanced), a surfactant, including anionic, non-ionic and ampholyticsurfactants, a hydrolase, building agents, bleaching agents, bluingagents and fluorescent dyes, caking inhibitors, solubilizers, cationicsurfactants and the like. All of these components are known in thedetergent art. The cellulase composition as described above can be addedto the detergent composition either in a liquid diluent, in granules, inemulsions, in gels, in pastes, and the like. Such forms are well knownto the skilled artisan. When a solid detergent composition is employed,the cellulase composition is preferably formulated as granules.Preferably, the granules can be formulated so as to contain a cellulaseprotecting agent. For a more thorough discussion, see U.S. Pat. No.6,162,782 entitled “Detergent compositions containing cellulasecompositions deficient in CBH I type components,” which is incorporatedherein by reference.

In yet another embodiment, the detergent compositions can also containenhanced levels of endoglucanase or altered endoglucanase. In thisregard, it really depends upon the type of product one desires to use indetergent compositions to give the appropriate effects.

Preferably the cellulase compositions are employed from about 0.00005weight percent to about 5 weight percent relative to the total detergentcomposition. More preferably, the cellulase compositions are employedfrom about 0.0002 weight percent to about 2 weight percent relative tothe total detergent composition.

Portions of the egl6 nucleic acid sequence that are capable of bindingto cellulose can be used to generate bacterial chimeric surfaceproteins, allowing whole-cell immobilization onto cellulose filters orother fibrous solid supports as described in Lehtio et al., 2001.

In addition the egl6 nucleic acid sequence finds utility in theidentification and characterization of related nucleic acid sequences. Anumber of techniques useful for determining (predicting or confirming)the function of related genes or gene products include, but are notlimited to, (A) DNA/RNA analysis, such as (1) overexpression, ectopicexpression, and expression in other species; (2) gene knock-out (reversegenetics, targeted knock-out, viral induced gene silencing (VIGS, seeBaulcombe, 1999); (3) analysis of the methylation status of the gene,especially flanking regulatory regions; and (4) in situ hybridization;(B) gene product analysis such as (1) recombinant protein expression;(2) antisera production, (3) immunolocalization; (4) biochemical assaysfor catalytic or other activity; (5) phosphorylation status; and (6)interaction with other proteins via yeast two-hybrid analysis; (C)pathway analysis, such as placing a gene or gene product within aparticular biochemical or signaling pathway based on its overexpressionphenotype or by sequence homology with related genes; and (D) otheranalyses which may also be performed to determine or confirm theparticipation of the isolated gene and its product in a particularmetabolic or signaling pathway, and help determine gene function.

Endoglucanases and beta-glucosidases may be responsible for theproduction of disaccharides, such as sophorose, fromcellooligosaccharides and glucose by transglycosylation reactions.Sophorose is known to be a very potent inducer of cellulase geneexpression (Ilmen, M. et al., 1997, Appl. Environ. Microbiol.63:1298-1306 and references therein). In this way EGs and BGLs may playan important role in the process of induction of cellulase geneexpression. Over-expression of certain EGs or BGLs in a fungal strainmay lead to higher overall cellulase productivity by that strain.

A. Homology to Known Sequences

The function of a related EGVI-encoding nucleic acid sequence may bedetermined by homology to known genes having a particular function. Forexample, a comparison of the coding sequence of an identified nucleicacid molecule to public nucleic acid sequence databases is used toconfirm function by homology to known genes or by extension of theidentified nucleic acid sequence.

The term “% homology” is used interchangeably herein with the term “%identity” herein and refers to the level of nucleic acid or amino acidsequence identity between the nucleic acid sequence that encodes EGVI orthe EGVI amino acid sequence, when aligned using a sequence alignmentprogram.

For example, as used herein, 80% homology means the same thing as 80%sequence identity determined by a defined algorithm, and accordingly ahomologue of a given sequence has greater than 80% sequence identityover a length of the given sequence. Exemplary levels of sequenceidentity include, but are not limited to, 80, 85, 90, 95, 98% or moresequence identity to a given sequence, e.g., the coding sequence foregl6, as described herein.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet athttp://www.ncbi.nim.nih.gov/BLAST/. See also, Altschul, et al., 1990 andAltschul, et al., 1997.

Sequence searches are typically carried out using the BLASTN programwhen evaluating a given nucleic acid sequence relative to nucleic acidsequences in the GenBank DNA Sequences and other public databases. TheBLASTX program is preferred for searching nucleic acid sequences thathave been translated in all reading frames against amino acid sequencesin the GenBank Protein Sequences and other public databases. Both BLASTNand BLASTX are run using default parameters of an open gap penalty of11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62matrix. (See, e.g., Altschul, et al., 1997.)

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-W program in MacVector version 6.5, operated with defaultparameters, including an open gap penalty of 10.0, an extended gappenalty of 0.1, and a BLOSUM 30 similarity matrix.

In one exemplary approach, sequence extension of a nucleic acid encodingegl6 may be carried out using conventional primer extension proceduresas described in Sambrook et al., supra, to detect egl6 precursors andprocessing intermediates of mRNA that may not have beenreverse-transcribed into cDNA and/or to identify ORFs that encode a fulllength protein.

In yet another aspect, the present invention includes the entire orpartial nucleotide sequence of the nucleic acid sequence of egl6 for useas a probe. Such a probe may be used to identify and clone outhomologous nucleic acid sequences from related organisms.

Screening of a cDNA or genomic library with the selected probe may beconducted using standard procedures, such as described in Sambrook etal., (1989). Hybridization conditions, including moderate stringency andhigh stringency, are provided in Sambrook et al., supra.

The probes or portions thereof may also be employed in PCR techniques togenerate a pool of sequences for identification of closely related egl6sequences. When egl6 sequences are intended for use as probes, aparticular portion of an EGVI encoding sequence, for example a highlyconserved portion of the coding sequence may be used.

For example, an egl6 nucleotide sequence may be used as a hybridizationprobe for a cDNA library to isolate genes, for example, those encodingnaturally-occurring variants of EGVI from other fungal, bacterial orplant species, which have a desired level of sequence identity to theegl6 nucleotide sequence disclosed in FIG. 1 (SEQ ID NO:1). Exemplaryprobes have a length of about 20 to about 50 bases.

B. Two Hybrid Analysis

Proteins identified by the present invention can be used in the yeasttwo-hybrid system to “capture” protein binding proteins which areputative signal pathway proteins. The yeast two hybrid system isdescribed in Fields and Song, Nature 340:245-246 (1989). Briefly, in atwo-hybrid system, a fusion of a DNA-binding domain-eg/6 (e.g.,GAL4-egl6 fusion) is constructed and transfected into yeast cells. Thewhole egl6 gene, or subregions of the egl6 gene, may be used. A secondconstruct containing the library of potential binding partners fused tothe DNA activation domain is co-transfected. Yeast co-transformantsharboring proteins that bind to the EGVI protein are identified by, forexample, beta-galactosidase or luciferase production (a screen), orsurvival on plates lacking an essential nutrient (a selection), asappropriate for the vectors used.

C. Microarray Analysis

In addition, microarray analysis, also known as expression profiling ortranscript profiling, may be used to simultaneously evaluate thepresence or expression of given DNA sequences, or changes in theexpression of many different genes. In one approach, a large set of DNAsequences (probes), usually a broad set of expressed sequence tags,cDNAs, cDNA fragments, or sequence-specific oligonucleotides, is arrayedon a solid support such as a glass slide or nylon membrane. Labelledtarget for hybridization to the probes is generated by isolating mRNAfrom control and induced tissue, then labeling each mRNA pool eitherdirectly or via a cDNA or cRNA intermediate, with a distinct marker,usually a fluorescent dye. The microarray is hybridized with the complexprobes, and the relative hybridization signal intensity associated witheach location on the array can be quantitated for each marker dye.Differences in expression between the control and induced states can bemeasured as a ratio of the signal from the two marker dyes. (SeeBaldwin, D et al., 1999.)

Microarray analysis of the source organism from which egl6 was derivedmay be carried out, to facilitate the understanding of gene function byidentifying other genes that are coordinately regulated as a consequenceof the overexpression of egl6. The identity of coordinately regulatedgenes may help to place the egl6 gene in a particular pathway.Alternatively, such analysis may be used to identify other genesinvolved in the same pathway using microarray analysis.

All publications, patents and patent applications are herein expresslyincorporated by reference in their entirety.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

EXAMPLE 1

In one exemplary approach, a cDNA fragment for use as a probe isisolated by extracting total RNA from mycelia of a T. reesei straingrown under conditions known to induce cellulase production andobtaining the polyadenylated (polyA) fraction therefrom. The polyA RNAis used to produce a cDNA pool which is then amplified using specificprimers based on the egl6 nucleic acid sequence provided herein.

Total RNA is isolated from the mycelia using methods known in the art,for example as described in Timberlake et al., 1981; Maniatis, et al.,1989; Ausubel, et al., 1993 and Sambrook et al., 1989, each of which isexpressly incorporated by reference herein. Once isolated, Northernblots are performed to confirm cellulase expression and select anoptimal induction time for cellulase expression and corresponding RNAisolation.

Messenger RNA (mRNA), having a poly (A) tail at the 3′ end, may bepurified from total RNA using methods known in the art.

The T. reesei RNA is used as template for RT-PCR using methods known inthe art (Loftus, J. et al., Science, 249:915-918, 1990). During thisprocedure the mRNA is reverse transcribed to produce first strand cDNA.The cDNA subsequently serves as template for PCR amplification of egl6cDNA sequences using specific olionucleotide primers designed inaccordance with SEQ ID No. 1 or SEQ ID No. 4. TABLE 1 Sequences ProvidedIn Support Of The Invention. SEQ. ID Description NO. full lengthT.reeseiegl6 cDNA nucleic acid 1 sequenceCCACGCGTCCGAGCAGTGTCCTCCTCCTCACTGCTTCGTCATGAAGGTCTCTCGAGTCCTTGCCCTTGTCCTGGGGGCCGTCATCCCTGCCCATGCTGCCTTTTCATGGAAGAACGTCAAGCTCGGCGGCGGCGGCGGCTTCGTCCCCGGCATCATCTTCCATCCCAAGACAAAAGGCGTAGCATATGCACGAACAGATATTGGCGGGCTGTACCGCCTCAACGCCGACGACTCATGGACCGCCGTCACGGATGGGATTGCTGATAATGCCGGCTGGCACAACTGGGGCATCGACGCTGTTGCGCTTGATCCGCAGGACGATCAAAAGGTGTATGCCGCAGTCGGCATGTATACGAACAGCTGGGATCCGAGTAATGGAGCCATCATTCGCTCGTCAGACCGCGGCGCAACGTGGTCCTTCACCAACTTGCCCTTCAAAGTCGGGGGTAACATGCCAGGACGCGGAGCCGGAGAGCGTCTGGCTGTCGATCCGGCCAACTCCAACATCATCTACTTTGGTGCTCGCTCAGGAAACGGCCTCTGGAAGTCTACGGACGGCGGCGTGACCTTTTCCAAGGTCTCGTCGTTCACGGCAACTGGGACGTACATCCCAGACCCGAGTGATTCCAACGGCTACAACAGCGACAAGCAAGGACTCATGTGGGTTACGTTCGACTCAACCAGCAGCACGACCGGGGGAGCCACGTCTCGTATCTTTGTTGGCACGGCTGATAACATCACTGCTTCAGTCTATGTGAGCACGAATGCCGGCTCCACGTGGAGTGCTGTACCGGGGCAGCCAGGGAAATACTTTCCTCACAAGGCGAAACTGCAGCCAGCAGAGAAGGCCTTGTATCTGACCTATTCCGATGGCACAGGGCCGTATGATGGCACACTTGGCTCAGTGTGGAGGTACGACATTGCAGGGGGAACTTGGAAAAGACATCACCCCTGTCTCTGGATCAGATCTATACTTTGGCTTTGGCGGCCTTGGCCTCGATTTGCAAAAGCCAGGAACCCTTGTTGTTGCTTCTTTGAACTCTTGGTGGCCAGATGCTCAGCTGTTTCGGTCGACCGACTCTGGGACAACATGGAGCCCGATCTGGGCGTGGGCGAGCTATCCGACTGAGACCTATTACTACAGCATCTCAACTCCCAAAGCACCGTGGATCAAGAACAACTTTATCGATGTGACGAGCGAGTCACCGTCCGATGGTCTCATCAAGCGCCTCGGCTGGATGATTGAGTCTCTCGAGATTGACCCAACCGACAGCAACCACTGGCTCTACGGCACCGGAATGACAATCTTTGGCGGCCACGATCTCACCAACTGGGACACGCGCCACAATGTGTCAATCCAATCACTGGCAGACGGCATCGAGGAATTCTCCGTCCAGGACCTGGCCTCTGCACCCGGCGGAAGCGAGCTATTGGCCGCAGTCGGAGACGACAACGGCTTCACCTTTGCCAGCAGAAACGACCTCGGGACATCGCCGCAGACGGTCTGGGCAACGCCCACATGGGCCACCTCGACGAGCGTCGACTACGCCGGGAACTCGGTCAAGAGCGTCGTCCGCGTCGGCAACACCGCCGGCACGCAACAGGTGGCCATCTCGTCCGACGGCGGCGCGACGTGGAGCATCGACTACGCGGCCGACACGTCCATGAACGGCGGCACGGTGGCCTATTCGGCCGACGGCGACACGATCCTCTGGTCGACCGCCTCGTCCGGCGTGCAGCGCTCGCAGTTCCAGGGCAGCTTTGCCTCCGTCTCGAGCCTGCCCGCGGGCGCCGTCATCGCCTCGGACAAGAAGACCAACAGCGTCTTCTACGCCGGCTCCGGATCGACCTTTTACGTCAGCAAGGACACCGGCAGCAGCTTCACGCGCGGGCCCAAGCTGGGCAGCGCAGGGACGATCCGGGATATCGCTGCTCACCCGACCACCGCGGGCACGTTGTATGTCTCGACCGACGTCGGCATATTCCGCTCCACAGACTCGGGCACGACCTTTGGCCAAGTCTCCACCGCCCTGACCAACACCTACCAGATCGCCCTGGGTGTGGGCTCAGGCTCGAACTGGAACCTGTATGCCTTCGGCACCGGCCCGTCAGGGGCTCGCCTCTACGCCAGTGGAGACAGCGGCGCCTCCTGGACGGACATCCAGGGCTCCCAGGGCTTCGGCTCCATCGACAGCACCAAGGTCGCCGGCAGCGGCAGCACCGCCGGGCAAGTCTACGTGGGCACCAACGGCCGGGGCGTCTTTTACGCTCAGGGAACCGTCGGCGGCGGCACGGGCGGGACTTCCTCGTCGACCAAGCAGAGCAGCAGCAGTACCTCTTCCGCCAGCTCGAGCACCACGCTGAGGTCGAGCGTTGTATCCACGACCCGGGCTTCGACGGTGACTTCGTCGAGGACCAGCTCGGCCGCCGGTCCCACGGGGTCAGGGGTCGCCGGTCATTATGCTCAGTGCGGAGGGATTGGGTGGACGGGGCCGACGCAGTGTGTGGCGCCGTATGTCTGCCAGAAGCAGAATGATTATTACTACCAGTGTGTGTGATGCTTGAACTGCCAAGCTCACGAGGAGAGCTACATACCCCTAGGCTCGCAGTAAAGAGCTCAAGCATCCGAAGAAGCACTAGTAGTAGAGATCCAGTCAGATAATTATCCATTTGTTTGAATTAAATGATCTTCTATTGAAA AAAAAAAAAAA T.reesei EGVIpredicted amino acid sequence 2AFSWKNVKLGGGGGFVPGIIFHPKTKGVAYARTDIGGLYRLNADDSWTAVTDGIADNAGWHNWGIDAVALDPQDDQKVYAAVGMYTNSWDPSNGAIIRSSDRGATWSFTNLPFKVGGNMPGRGAGERLAVDPANSNIIYFGARSGNGLWKSTDGGVTFSKVSSFTATGTYIPDPSDSNGYNSDKQGLMWVTFDSTSSTTGGATSRIFVGTADNITASVYVSTNAGSTWSAVPGQPGKYFPHKAKLQPAEKALYLTYSDGTGPYDGTLGSVWRYDIAGGTWKDITPVSGSDLYFGFGGLGLDLQKPGTLVVASLNSWWPDAQLFRSTDSGTTWSPIWAWASYPTETYYYSISTPKAPWIKNNFIDVTSESPSDGLIKRLGWMIESLEIDPTDSNHWLYGTGMTIFGGHDLTNWDTRHNVSIQSLADGIEEFSVQDLASAPGGSELLAAVGDDNGFTFASRNDLGTSPQTVWATPTWATSTSVDYAGNSVKSVVRVGNTAGTQVAISSDGGATWSIDYAADTSMNGGTVAYSADGDTILWSTASSGVQRSQFQGSFASVSSLPAGAVIASDKKTNSVFYAGSGSTFYVSKDTGSSFTRGPKLGSAGTIRDIAAHPTTAGTLYVSTDVGIFRSTDSGTTFGQVSTALTNTYQIALGVGSGSNWNLYAFGTGPSGARLYASGDSGASWTDIQGSQGFGSIDSTKVAGSGSTAGQVYVGTNGRGVFYAQGTVGGGTGGTSSSTKQSSSSTSSASSSTTLRSSVVSTTRASTVTSSRTSSAAGPTGSGVAGHYAQCGGIGWTGPTQCVAPYVCQKQ NDYYYQCV T.reesei EGVIprotein predicted signal 3 sequence: MKVSRVLALVLGAVIPAHA T.reesei egl6nucleic acid coding sequence 4ATGAAGGTCTCTCGAGTCCTTGCCCTTGTCCTGGGGGCCGTCATCCCTGCCCATGCTGCCTTTTCATGGAAGAACGTCAAGCTCGGCGGCGGCGGCGGCTTCGTCCCCGGCATCATCTTCCATCCCAAGACAAAAGGCGTAGCATATGCACGAACAGATATTGGCGGGCTGTACCGCCTCAACGCCGACGACTCATGGACCGCCGTCACGGATGGGATTGCTGATAATGCCGGCTGGCACAACTGGGGCATCGACGCTGTTGCGCTTGATCCGCAGGACGATCAAAAGGTGTATGCCGCAGTCGGCATGTATACGAACAGCTGGGATCCGAGTAATGGAGCCATCATTCGCTCGTCAGACCGCGGCGCAACGTGGTCCTTCACCAACTTGCCCTTCAAAGTCGGGGGTAACATGCCAGGACGCGGAGCCGGAGAGCGTCTGGCTGTCGATCCGGCCAACTCCAACATCATCTACTTTGGTGCTCGCTCAGGAAACGGCCTCTGGAAGTCTACGGACGGCGGCGTGACCTTTTCCAAGGTCTCGTCGTTCACGGCAACTGGGACGTACATCCCAGACCCGAGTGATTCCAACGGCTACAACAGCGACAAGCAAGGACTCATGTGGGTTACGTTCGACTCAACCAGCAGCACGACCGGGGGAGCCACGTCTCGTATCTTTGTTGGCACGGCTGATAACATCACTGCTTCAGTCTATGTGAGCACGAATGCCGGCTCCACGTGGAGTGCTGTACCGGGGCAGCCAGGGAAATACTTTCCTCACAAGGCGAAACTGCAGCCAGCAGAGAAGGCCTTGTATCTGACCTATTCCGATGGCACAGGGCCGTATGATGGCACACTTGGCTCAGTGTGGAGGTACGACATTGCAGGGGGAACTTGGAAAGACATCACCCCTGTCTCTGGATCAGATCTATACTTTGGCTTTGGCGGCCTTGGCCTCGATTTGCAAAAGCCAGGAACCCTTGTTGTTGCTTCTTTGAACTCTTGGTGGCCAGATGCTCAGCTGTTTCGGTCGACCGACTCTGGGACAACATGGAGCCCGATCTGGGCGTGGGCGAGCTATCCGACTGAGACCTATTACTACAGCATCTCAACTCCCAAAGCACCGTGGATCAAGAACAACTTTATCGATGTGACGAGCGAGTCACCGTCCGATGGTCTCATCAAGCGCCTCGGCTGGATGATTGAGTCTCTCGAGATTGACCCAACCGACAGCAACCACTGGCTCTACGGCACCGGAATGACAATCTTTGGCGGCCACGATCTCACCAACTGGGACACGCGCCACAATGTGTCAATCCAATCACTGGCAGACGGCATCGAGGAATTCTCCGTCCAGGACCTGGCCTCTGCACCCGGCGGAAGCGAGCTATTGGCCGCAGTCGGAGACGACAACGGCTTCACCTTTGCCAGCAGAAACGACCTCGGGACATCGCCGCAGACGGTCTGGGCAACGCCCACATGGGCCACCTCGACGAGCGTCGACTACGCCGGGAACTCGGTCAAGAGCGTCGTCCGCGTCGGCAACACCGCCGGCACGCAACAGGTGGCCATCTCGTCCGACGGCGGCGCGACGTGGAGCATCGACTACGCGGCCGACACGTCCATGAACGGCGGCACGGTGGCCTATTCGGCCGACGGCGACACGATCCTCTGGTCGACCGCCTCGTCCGGCGTGCAGCGCTCGCAGTTCCAGGGCAGCTTTGCCTCCGTCTCGAGCCTGCCCCGCGGGCGCCGTCATCGCCTCGGACAAGAAGACCAACAGCGTCTTCTACGCCGGCTCCGGATCGACCTTTTACGTCAGCAAGGACACCGGCAGCAGCTTCACGCGCGGGCCCAAGCTGGGCAGCGCAGGGACGATCCGGGATATCGCTGCTCACCCGACCACCGCGGGCACGTTGTATGTCTCGACCGACGTCGGCATATTCCGCTCCACAGACTCGGGCACGACCTTTGGCCAAGTCTCCACCGCCCTGACCAACACCTACCAGATCGCCCTGGGTGTGGGCTCAGGCTCGAACTGGAACCTGTATGCCTTCGGCACCGGCCCGTCAGGGGCTCGCCTCTACGCCAGTGGAGACAGCGGCGCCTCCTGGACGGACATCCAGGGCTCCCAGGGCTTCGGCTCCATCGACAGCACCAAGGTCGCCGGCAGCGGCAGCACCGCCGGGCAAGTCTACGTGGGCACCAACGGCCGGGGCGTCTTTTACGCTCAGGGAACCGTCGGCGGCGGCACGGGCGGGACTTCCTCGTCGACCAAGCAGAGCAGCAGCAAGTACCTCTTCCGCCAGCTCGAGCACCACGCTGAGGTCGAGCGTTGTATCCACGACCCGGGCTTCGACGGTGACTTCGTCGAGGACCAGCTCGGCCGCCGGTCCCACGGGGTCAGGGGTCGCCGGTCATTATGCTCAGTGCGGAGGGATTGGGTGGACGGGGCCGACGCAGTGTGTGGCGCCGTATGTCTGCCAGAAGCAGAATGATTATTACTACCAGTGTGTGTGA

1-26. (canceled)
 27. A method of producing ethanol, said methodcomprising the steps of: a) contacting a biomass composition with anenzymatic composition comprising endoglucanase 6 to yield a sugarsolution; b) adding to the sugar solution a fermentative microorganism;and c) culturing the fermentative microorganism under conditionssufficient to produce ethanol, wherein the biomass composition may beoptionally pretreated.
 28. The method of claim 27 wherein step (a)further comprises the addition of at least one endoglucanase.
 29. Themethod of claim 27 wherein step (a) further comprises the addition of atleast one cellbiohydrolase.
 30. The method of claim 28 wherein step (a)further comprises the addition of at least one cellbiohydrolase.
 31. Themethod of claim 27 wherein the pretreatment is with a dilute acid.
 32. Amethod of producing ethanol, said method comprising the steps of: a)contacting a biomass composition with an enzymatic compositioncomprising a β-glucosidase 4 and a fermentative microorganism; and b)culturing the fermentative microorganism under conditions sufficient toproduce ethanol, wherein the biomass composition may be optionallypretreated.
 33. The method of claim 32 wherein step (a) furthercomprises the addition of at least one endoglucanase.
 34. The method ofclaim 32 wherein step (a) further comprises the addition of at least onecellbiohydrolase.
 35. The method of claim 33 wherein step (a) furthercomprises the addition of at least one cellbiohydrolase.
 36. The methodof claim 32 wherein the pretreatment is with a dilute acid.